Effects of short‐ and long‐term use of propolis extracts on liver and kidney in rats

Abstract Propolis is widely used as a supplementary food product for its health benefits. The aim of this study was to determine the effects of commercial propolis extracts on the liver and kidney. Propolis extracts (250 mg/kgbw/day) were administered orally to adult male Wistar albino rats in solvents of ethanol, propylene glycol, water, and olive oil. Liver enzyme levels were determined biochemically in blood samples, and histopathological examinations were performed on the liver. Damage rate in both kidney tissue in the propolis‐ethanol extract group increased significantly compared with the other groups after 30 and 90 days of application (p < .05). According to the results, ethanol, used as a common solvent in propolis products, may adversely affect the liver in long‐term use. The data indicate that propolis‐olive oil extract may be an essential alternative due to its effective and reliable properties.

In recent years, due to the potential for the development of new medicines, propolis has been extensively studied.It has immunomodulatory, anti-inflammatory, antioxidant, antibacterial, antiviral, antifungal, and antiparasitic bioactivity (Sforcin & Bankova, 2011).
The extraction must take place with solvents, as raw propolis is not suitable for consumption (Pietta et al., 2002).Solvents in propolis extraction are divided into two groups organic (water and oil) and alcoholic (ethyl alcohol, propylene glycol, glycol, glycerol, etc.).The antimicrobial characteristics of the extract, extraction yield, and content of phenolic and flavonoid substances can be influenced by the propolis extraction method (Pobiega et al., 2019).Propolis extraction with alcohol is a simple and effective method (Silici & Baysa, 2020).The ethanolic extract of propolis (EEP) is a rich source of phenolic acids and flavonoids.Studies confirm that the extraction of propolis with ethanol has immunomodulatory, chemo-preventive, and antitumor effects (Sforcin, 2007).Even though there are beneficial effects of EEP on health, it can also show toxic effects (Bonamigo et al., 2017).
Ethyl alcohol, which is a commonly used solvent in propolis extraction, is rapidly absorbed from the gastrointestinal tract upon oral ingestion and distributed throughout body fluids.The rate of distribution of ethyl alcohol in body fluids and tissues depends on the size, permeability, and blood flow of the tissue.Ethyl alcohol is mainly (90%) metabolized in the liver.The main pathway in the biotransformation of ethyl alcohol is its oxidation in the liver to acetaldehyde and hydrogen.In addition, ethanol and its metabolites are excreted in the urine, and its content in urine is higher than in blood and liver.
Chronic alcohol administration reduces renal tubular reabsorption and renal function.Functional abnormalities of renal tubules may be associated with ethanol-induced changes.For these reasons, we preferred both enzymatic and histological examination in liver tissue and histological examination in kidney.
Liver enzymes may be increased by prolonged use of propolis (D'Ercole, 2020).Chronic ethanol consumption harms hepatocytes through oxidative stress and lipid oxidation (Diehl, 2005).Like the liver, kidneys express cytochrome P450 2E1 enzyme, which catabolizes ethanol with free radical formation (Latchoumycandane et al., 2014).Therefore, ethanol catabolism can also damage the kidneys.Sudden loss of kidney function in patients with alcoholic hepatitis is an indicator of an increased risk of mortality (Altamirano et al., 2012).Although the daily consumption of propolis-ethanol extract is low, this risk can pose liver and kidney health in long-term use.Furthermore, the use of EEP in cancer and diabetes patients and children is limited.For this reason, researchers have been in search of more harmless extraction solvents (Sforcin, 2016).
In this study, we aimed to reveal the short-and long-term effects of commonly used commercial propolis extracts on liver and kidney tissues in rats.Liver enzymes such as gamma-glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) and liver and kidney tissue histology were examined to determine these effects.

| Animals
Ten separate groups were randomly selected from a total of 80 adult male Wistar albino rats (8 weeks age, 240-280 g).For 30 and 90 days, oral gavage was used to give ethanol, olive oil, propylene glycol, and a water-based propolis extract (each dose individually) (Eraslan et al., 2008).The rats were housed in cages, fed with standard commercial pellet food, and maintained in a regular laboratory environment with 12-h light/dark cycles.Feed and water were given ad libitum.After the experiment, rats were given ketamine/xylazine anesthesia to minimize the pain of sacrifice while having blood samples extracted from the heart.Ethics committee approval was obtained from Erciyes University Animal

Experiments Local Ethics Committee (Ethics Committee Approval
No: 04.03.2020-20/063) for the study.There were eight rats in each group.

| Propolis
The propolis sample used in the research was prepared by Erciyes University Technopark (Nutral Therapy).The botanical origin of the propolis used in this study is black poplar (Populus nigra L.) and its geographical origin is Kayseri (Central Anatolia, Turkey).For extraction, raw propolis samples were ground and homogenized.Twenty-five grams of propolis powder was extracted from 100 mL of ethanolwater (70:30), distilled water, olive oil, and propylene glycol (PG), respectively.The ratio of the solid material to the solvents was 4:10.
Ultrasound extraction was conducted according to the method of Netíková et al. (2013) with some modifications.Briefly, the solidsolvent mixtures were shaken for 24 h at 20°C using a shaking water bath.Then ultrasound-assisted (UA) and conventional (C) extracts were centrifuged at 4427× g for 10 min.The extracts were then filtered through a Whatman No. 1 filter paper and stored at +4°C.

| Analysis of blood samples
Blood samples were collected from the animals only by the end of the 30 and 90 days of the study.The animals were fasted for 6 h before blood samples were taken.General anesthesia was not given before the samples were taken since there was a chance that the biochemical parameters may alter.Just prior to taking blood samples, the animals were kept under a light ether anesthetic.Blood samples were then slowly taken from each animal into tubes both with and without anticoagulants using a cannula placed in the heart.Blood was taken from every eight animals in each group.Blood samples were collected into both types of tubes, and analyses for the parameters under investigation were done on the same day.Utilizing a Konelab 60i model auto-analyzer and a kit from the same manufacturer, serum biochemical parameters including GGT, LDH, AST, ALT, and ALP were measured using Johnson & Johnson Label kits (Eraslan et al., 2009).

| Histological analysis
The tissues collected at the end of the experiment were fixed with 10% formalin for 1 week.The tissues were then dehydrated through 50%, 70%, 80%, 96%, and 100% ethanol, cleared with xylene, and embedded in paraffin wax.Sections were cut at 5 μm and stained with Harris Hematoxylin Eosin, mounted with Entellan (Merck, Darmstadt, Germany) to slides.Olympus, Tokyo, Japan's BX53 light microscope was used to analyze the preparations (Doǧanyiǧit et al., 2020).In the kidney sections, enlargement of the tubular lumen, glomerular degeneration, vacuolization, bleeding, and shedding of cells in the tubule were examined and tubular debris was assessed.Dilated blood vessels, inflammatory cell infiltration, and damaged hepatocytes with pycnotic nuclei were evaluated in the liver sections.Histopathological results in each category were scored as "0 = absent, 1 = mild, 2 = moderate, and 3 = severe" (Doǧanyiǧit et al., 2020;Inandiklioglu et al., 2021).Quantification was performed by two investigators blindly.

| Statistical analysis
Biochemical data were presented as mean ± standard error of mean (SEM).Statistical Package for Social Sciences (SPSS) version 22.0 for Windows (SPSS Inc., Chicago, IL) was used to compare data across groups using one-way analysis of variance (ANOVA) and post hoc Tukey honestly significant difference (HSD) tests were conducted.
Histopathological score data expressed as ±SEM (standard error of the mean).Two-way ANOVA (analysis of variance), Sidak's multiple comparison test (to examine the difference between 30 and 90 days of application) and TUKEY's multiple comparison test (to compare 30-day and 90-day treatment groups within themselves) were applied.

| Histological analysis in kidney tissue of experimental groups
The effects of ethanol-, propylene glycol-, water-, and olive oil-based extracts of propolis on kidney tissue were histologically evaluated for 30 and 90 days.As shown in Figure 1a, kidney images of control groups exhibited normal histological structure.Especially in the propolis-ethanol group, vacuolization, and bleeding were observed in 30 days of application.In the 90-day practice, the enlargement of the tubular lumen, bleeding, and the residual materials pouring into the tubule were concentrated.When we compare the 90-day treatment groups, propolis-ethanol group's damage rate analyzed in these groups significantly increased compared to the other groups (*p < .05).As we examined the difference between the amount of damage after 30 and 90 days of application in the groups, only the propolis-ethanol group's damage rate at the end of the 90 days significantly increased compared with the 30 days (*p < .05)(Figure 1b).

| Histological analysis in liver tissue of experimental groups
The effects of ethanol-, propylene glycol-, water-, and olive oil-based extracts of propolis on liver in rats were histologically evaluated during 30 and 90 days.As shown in Figure 2a, liver images belonging to the control groups exhibited normal histological structure.
Inflammatory cell infiltration and dilated blood vessels increased in liver sections of rats treated with propolis-water especially at the end of 30 days.The incidence of tissue damage decreased in the groups that were applied propolis-water for 90 days.As the propylene-glycol group was applied for 90 days, inflammatory cells increased, and sinusoidal dilatation was increased when compared with the group applied for 30 days (*p < .05).Bleeding was observed in the 30-day application in the propolis-ethanol group.In 90 days of application, bleeding decreased but inflammatory cells increased, and sinusoidal dilatation increased (*p < .05).When the other groups were compared in days, the rate of damage in the short-and longterm applications was found to be similar (*p < .05)(Figure 2b).
When we examine the difference between the amount of damage after 30 and 90 days of application in the groups, only the WEP group damage rate after 90 days was significantly lower than at the end of 30 days (*p < .05).

| Biochemical findings
The effect of ethanol-, propylene glycol-, water-, and olive oil-based propolis application on the levels of liver enzymes AST, ALT, ALP, LDH, and GGT in blood samples was evaluated.At the end of 1 month application, AST levels increased in EEP, PGEP, and WEP groups compared with the control group, but this change was close to the control group in the OOEP group (Figure 3a).In ALT results, the EEP and PGEP groups were higher than the control (Figure 3b).As the LDH levels in blood were evaluated, the EEP group significantly increased ( # p < .05)compared with the control group (Figure 3d).
AST results showed a significant increase in ethanol and propylene groups after 3 months of application (*p < .05).In ALT results, the ethanol group significantly increased compared with control and other groups (*p < .05).As the LDH results were examined, the increase in the EEP and PGEP groups was significant compared with the other groups (*p < .05).Expressed as ±SEM (standard error of the mean).Two-way analysis of variance (ANOVA) and (to examine the difference between 30 and 90 days of application) and TUKEY's multiple comparison test (to compare 30-day and 90-day application groups) were applied (*p < 0.0001 represents statistically significant difference).
F I G U R E 3 Biochemical analysis of liver.The effects of olive oil, water, propylene glycol and ethanol extract of propolis on aspartate transaminase (a), alanine transaminase (b), alkaline phosphatase (c), lactate dehydrogenase (d), and glutamyl transpeptidase (e) levels in blood samples were examined.Biochemical enzyme values shown in histogram graph.Expressed as ±SEM (standard error of the mean).Two-way analysis of variance (ANOVA) (to examine the difference between 30 and 90 days of application) and TUKEY's multiple comparison test (to compare 30-day and 90-day treatment groups) were applied (ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gammaglutamyl transpeptidase; LDH, lactate dehydrogenase; # p < .05represents that it is different from the control group in the 30-day application groups, *p < .05represents that it is different from the control group in the 90-day application groups).When all results are evaluated, enzyme activities in ethanol groups significantly increased (*p < .05),followed by PGEP and WEP groups, respectively, in the 1-month and 3-month applications.
Hematological findings in rats given different propolis extracts for 90 days, AST, ALP, ALT, and LDH values were significantly different from each other.

| DISCUSS ION
In our study, we evaluated the histological changes, damage rates, and plasma levels of ALT, AST, ALP, LDH, and GGT enzymes to demonstrate the effect of solvents used in propolis extraction on liver and kidney tissues.We analyzed the differences in the control, OOEP, WEP, EEP, and PGEP groups through oral gavage administration of extracts.
Hepatoprotective and nephroprotective properties have been demonstrated for the active component of poplar-type propolis, caffeine acid phenethyl ester (CAPE).A 15-day study on rats showed that chestnut propolis has a protective effect against the oxidative damage caused by alcohol (Kolankaya et al., 2002).Another study demonstrated that CAPE protects from necrosis, lipid peroxidation, p65 activation, and abnormal cell proliferation (Macias-Perez et al., 2013).Additionally, it was observed that preapplication with a single dosage of CAPE reduced the incidence of hepatic tumors caused by diethylnitrosamine (DEN) by 43% and that CAPE protects against aflatoxin B1-induced hepatotoxicity through the control of free radical generation (Akcay et al., 1994;Beltrán-Ramírez et al., 2012).
Furthermore, it was discovered that CAPE reduced systemic inflammation brought on by LPS and galactosamine in rats, which reduced damage to hepatic and neural cells (Korish & Arafa, 2011).
Additionally, hepatic steatosis brought on by a high-fat diet in a mouse model was seen to be improved by CAPE treatment.This improvement was associated with reductions in c-N-terminal kinase and nuclear factor kappa-B (NF-kB) activation as well as decreased expression of cyclooxygenase (COX) 22 (Bezerra et al., 2012).In a study utilizing electron microscopy, it was demonstrated that CAPE protects hepatocytes against ultrastructural changes induced by cholesterol (Esrefoglu et al., 2012).The antioxidant activity of CAPE has been shown to reduce oxidative damage in the liver, as well as mitigate cytokine damage and necroinflammation in rats with biliary bleeding.In addition, preadministration of CAPE significantly prevented tamoxifen-induced liver toxicity in rats, attributed to the attenuation of antioxidant lipid peroxidation and the restoration of enzyme activity (Albukhari et al., 2009).While CAPE has demonstrated hepatoprotective and nephroprotective effects, its longterm benefits rely on appropriate pairing with the right extract.
The protective effects of EEP on alcohol-induced fatty liver in rats were investigated, and 4-week EEP treatment showed a restoration in liver enzyme levels (Ye et al., 2019).The oral administration of EEP for a duration of 7 days has been observed to upregulate the expression of genes crucial for the antioxidant pathway in the livers of mice (Hotta et al., 2020).These findings suggest that EEP may serve as a potential therapeutic agent to prevent diseases associated with oxidative stress.We investigated the effects of an ethanol extract of propolis on the liver over 30 and 90 days of application.The amount of damage to liver tissue significantly increased in the EEP group.Moreover, we observed elevated levels of liver enzymes AST, ALT, and LDH as a result of EEP application.Therefore, we postulate that prolonged EEP usage can damage the liver tissue, whereas its short-term application exhibits liver protective effect.
EEP affects the kidney as well as the liver tissue.Susanto roles in chronic kidney disease (CKD).In experimental nephropathies, anti-inflammatories and antioxidants significantly protect the kidneys.Red propolis from Brazil was used to create a kidney ablation model.When propolis was given to rats with significant proteinuria and hypertension 30 days after surgery, significant declines in glomerulosclerosis, proteinuria, serum creatinine retention, glomerulosclerosis, renal macrophage infiltration, and oxidative stress were observed 90 days later (Teles et al., 2015).
In conclusion, propolis treatment reduced hypertension and structural kidney damage in the tested model.Baykara et al. (2015) showed that creatine urea, MDA, GSH, SOD, and GSH-Px levels increased in rats resulting from the administration of contrast agent (Diatrizoate), and propolis normalized these values as effectively as N-acetyl cysteine and showed nephroprotective activity.
Chronic kidney disease can develop as a result of damaged tubular epithelial cells secreting pro-inflammatory cytokines and profibrotic substances (Geng et al., 2012;Liu et al., 2018;van Kooten et al., 1999;Yard et al., 1992).Accordingly, chronic use of EEP may damage the renal tubules, causing serious kidney diseases such as CKD.
Another solvent tested in propolis extracts is propylene glycol in this study.We investigated the effects of PGEP on the liver and kidney.In our study, the increase in exposure time to PGEP significantly increased the damage rate in both kidney and liver tissue.In addition, the rate of kidney and liver damage at the end of 30 days in the propylene glycol group increased compared to the control and olive oil groups.In this group, the increase in ALT, AST, and LDH enzyme levels at the end of 90 days is indicative of liver damage.A study investigating the pharmacokinetics of propylene glycol showed that kidney's ability to excrete propylene glycol declines at large concentrations (Speth et al., 1987).Accordingly, 3-month PGEP was more harmful compared with 1 month may be related to the decrease in kidney excretion.Propylene glycol is generally considered to be nontoxic.However, excessive propylene glycol use causes metabolic acidosis and acute kidney damage (Bruns et al., 1982).On the other hand, PGEP has been found to suppress bacterial growth and biofilm production (Meto et al., 2020).Therefore, propolis is envisioned as a valuable source of natural compounds for the development of new therapeutic strategies against biofilm-associated infections.These studies indicate that although PGEP has a beneficial effect, excessive and prolonged use can cause toxicity.
According to the study on the effects of WEP, it was observed that WEP has protective effects against influenza A virus and ultraviolet rays (Saito et al., 2015;Takemura et al., 2012).As compared to EEP, WEP has higher antioxidant activity and is effective for most harmful microorganisms.Using microkernel experiments, the use of WEP has been shown to be safe (Rocha et al., 2013).However, in our study, the amount of damage to the liver tissue significantly increased after the long-term WEP application.Histochemical examination revealed that enlarged blood vessels and inflammatory cell infiltration increased in the liver at the end of 30 days.Similarly, another study found that WEP can suppress mitochondrial respiration in the heart (Majiene et al., 2006).Therefore, we assume that WEP can damage liver and heart tissue.Although water is a natural solvent, the use of chemicals to increase water solubility can pose a risk for WEP.
Efficacy of different propolis extracts was investigated in the literature.Propolis extracts using oil, ethanol, and propylene glycol all exhibited antibacterial and antifungal activities.However, the antimicrobial effect of glycerol solution was only temporary, lasting for a few days (Tosi et al., 1996).Researchers investigated the flavonoid content of crude propolis produced by Trigona bees in Indonesia (Sulawesi region) by extracting it with various solvents (water, ethanol, propylene glycol, olive oil, and coconut oil).They found that using oil as a solvent for propolis extraction resulted in comparable flavonoid content to that of the ethanol extract.Both olive oil and coconut oil were identified as suitable solvents for propolis extraction (Pujirahayu et al., 2014).In a study comparing various antioxidants, OOEP at 0.01% levels exhibited superior antioxidant activity than butylated hydroxyanisole and butylated hydroxytoluene.The highest antioxidant activity was observed at 0.08% concentration.The researchers concluded that antioxidant activity of propolis increased with higher concentrations and suggested that olive oil extract could serve as a natural antioxidant source (Özcan, 2000).The properties of the poplar-type OOEP were investigated, focusing on its antioxidant, antiradical, and antipyretic effects, along with its total phenolic content.Increasing propolis concentration was found to enhance total phenolic content, antioxidant, and antiradical activity.
Moreover, the olive oil extract of propolis demonstrated antipyretic effects in rats with yeast-induced hyperthermia.These findings suggest that OOEP could serve as a healthier alternative to alcoholic extracts, displaying beneficial antioxidant, antiradical, and antipyretic effects (Silici & Baysa, 2020).

| CON CLUS ION
The biological effects of propolis extractions in long-term use have not been adequately studied.Therefore, we examined effects of ethanol, propylene glycol, olive oil, and water extracts of propolis on kidney and liver tissues in short and long-term use.Our study did not reveal any significant difference in the amount of damage to kidney and liver tissues in the propolis-olive oil group compared with the control group.We postulate that olive oil-based propolis extract could serve as a healthier substitute for alcoholic extracts.
Moreover, the use of olive oil extract was thought to be safer than ethanol and propylene glycol extracts.In our biochemical analysis, a significant increase in AST, ALT, and LDH enzymes was interpreted as a result of ethanol extract use.Damage to the liver and kidney tissues of male adult Wistar rats may be associated with prolonged use

ACK N OWLED G M ENTS
None.

FU N D I N G I N FO R M ATI O N
This research was supported by the Erciyes University BAP (Scientific Research Projects) Unit (grant no.FBA 2020-10528).

CO N FLI C T O F I NTE R E S T S TATE M E NT
There is no conflict of interest among the authors.

F
Histopathological analysis of the kidney.(a) Hematoxylin eosin staining images of rat kidney tissues belonging to the experimental groups (star: glomerular degeneration, green arrow: bleeding, yellow arrow: enlargement of the tubular lumen, thin yellow arrow: vacuolization, blue arrow: tubular debris).(b) *p < 0.0001 represents statistically significant difference.F I G U R E 2 Histopathological analysis of the liver.(a) Hematoxylin eosin staining images of rat liver tissues belonging to the experimental groups (thick blue arrow: sinusoidal dilatation; thin blue arrow: inflammatory cell infiltration and asterisk: bleeding).(b) Histopathological score data shown in the histogram graph.
et al. (2018) investigated the nephroprotective effect of propolis (50 mg/kg) on interstitial fibrosis, systolic blood pressure, and body weight in mice with unilateral ureteral obstruction.As a result, propolis increased body weight by decreasing interstitial fibrosis and systolic blood pressure.Due to its ability to absorb and concentrate hazardous substances along with its high blood flow, the kidney is the target of toxic compounds.Azab et al. (2014) assessed the effectiveness of various natural compounds (curcumin, rosemary, and propolis) against gentamicin-induced nephrotoxicity and histological and biochemical alterations in mice.In addition to greatly reducing the raised blood levels of urea, creatinine, and uric acid, co-administration of curcumin, rosemary, and propolis significantly contributed to the detrimental structural alterations in the kidney brought on by gentamicin.In another study,Osman and Hafez Tantaway (2013) investigated propolis's total phenol and flavonoid content, in vitro antioxidant activity, and potential anti-nephrotoxic properties in rabbits.Utilizing biochemical indicators (measured of blood urea and creatinine) and histological kidney alterations, propolis' protective effects on gentamicin-induced nephrotoxicity in rabbits were assessed.In conclusion, gentamicin's histopathological and biochemical side effects were dramatically reduced after oral administration of the propolis.The kidney is a crucial organ that is important in the detoxification and elimination of xenobiotics.Therefore, it is crucial to study natural kidney-protecting substances.Aldamash et al. (2016) tested the renoprotective effects of propolis against gentamicin-induced renal toxicity in mice.With the administration of gentamicin, collagen, and reticular fibers were detected by histochemistry, and immunohistochemistry revealed an increase in kidney injury (Kim-1 gene expression), oxidative stress (MDA gene expression), and apoptosis (caspase-3 gene expression).Propolis and gentamicin co-administration results in a significant reduction in BUN levels, a reduction in tubular damage, the appearance of healthy glomeruli with normal cellularity, a reduction in collagen and reticular fiber deposition, and a reduction in apoptosis, kidney damage, and oxidative stress.These results demonstrate the kidney-protective role of propolis against gentamicin-induced toxicity.Inflammation and oxidative stress both play pathogenic of ethanol extract of propolis.As a result, attention should be paid to the dose and duration of use as well as the solvent used in the extraction of propolis to protect and support health.AUTH O R CO NTR I B UTI O N S Sibel Silici: Conceptualization (lead); data curation (equal); formal analysis (equal); funding acquisition (lead); investigation (lead); methodology (equal); project administration (lead); resources (equal); supervision (equal); validation (equal); visualization (equal); writing -original draft (equal); writing -review and editing (equal).Sevim Demiray: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); methodology (equal); resources (equal); validation (equal); visualization (equal); writingoriginal draft (equal); writing -review and editing (equal).Aslı Okan: Data curation (equal); investigation (equal); methodology (equal); resources (equal); validation (equal); writing -original draft (equal); writing -review and editing (equal).Sena Ertuğrul: Data curation (equal); investigation (equal); methodology (equal); resources (equal); validation (equal); writing -original draft (equal); writing -review and editing (equal).Sahar Alizada: Conceptualization (equal); data curation (equal); methodology (equal); resources (equal); validation (equal); writing -original draft (equal); writing -review and editing (equal).Züleyha Doğanyiğit: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); investigation (equal); methodology (equal); project administration (equal); resources (equal); supervision (equal); validation (equal); visualization (equal); writing -original draft (equal); writing -review and editing (equal).